This invention relates to the use of cell-division controlling proteins or parts thereof, preferably cell-division controlling proteins that bind retinoblasoma-like proteins, more preferably cyclins, particularly D-type cyclins and genes encoding same, for producing plants with modified phenotypes, particularly plants with modified growth rates or plants comprising parts with modified growth rates and/or modified relative sizes or plants with modified architecture. This invention also relates to plant cells and plants expressing such DNAs.
All eukaryotic cells undergo the same sequential series of events when they divide, and the term xe2x80x9ccell cyclexe2x80x9d reflects the ordered nature and universality of these events. In the eukaryotic cell cycle DNA replication (S) and cell division (M) are normally temporally separated by xe2x80x9cgapxe2x80x9d phases (G1 and G2) in the sequence G1-S-G2-M. This arrangement allows entry to the critical processes of DNA replication and mitosis to be precisely controlled. Underlying the cytological events of the cell cycle is an ordered series of temporally and spatially organised molecular and cellular processes which define the direction and order of the cycle. Cell cycle progression appears to be regulated in all eukaryotes by major controls operating at the G1-to-S phase and G2-to-M phase boundaries. Passage through these control points requires the activation of cyclin-dependent kinases (CDKs), whose catalytic activity and substrate specificity are determined by specific regulatory subunits known as cyclins and by interactions with other proteins that regulate the phosphorylation state of the complex (reviewed in Atherton-Fessier et al., 1993; Solomon, 1993). In budding and fission yeasts, both the G1-to-S and G2-to-M phase transitions are controlled by a single CDK, encoded by the cdc2+ gene in Schizosaccharomyces pombe and by CDC28 in Saccharomyces cerevisiae. The association of p34cdc2 (p34CDC28 in budding yeast) with different cyclin partners distinguishes the two control points (reviewed in Nasmyth, 1993). In mammalian cells, a more complex situation prevails, with at least six related but distinct CDKs (encoded by cdc2/cdk1, cdk2, cdk3, cdkain 4, cdk5, and cdk6) having distinct roles, each in conjunction with one or more cognate cyclin partners (Fang and Newport, 1991; Meyerson et al., 1991, 1992; Xiong et al., 1992b; Tsai et al., 1993a; van den Heuvel and Harlow, 1993; Meyerson and Harlow, 1994). B-type cyclins are the major class involved in the G2-to-M transition and associate with p34cdc2 or its direct homologs (reviewed in Nurse, 1990). Cyclin B is one of two cyclins originally described as accumulating in invertebrate eggs during interphase, and rapidly destroyed in mitosis (Evans et al., 1983), and it is a component of Xenopus maturation-promoting factor (Murray et al., 1989). Subsequently, cyclin B homologs have been identified from many eukaryotic species. Cyclin A is also of widespread occurrence in multicellular organisms, and its precise role is unclear, although its peak of abundance suggests a function in S phase (reviewed in Pines, 1993).
The G1-to-S phase transition is best understood in S. cerevisiae. Genetic studies define a point late in G1 called START. After passing START, cells are committed to enter S phase and to complete a full additional round of division, which will result in two daughter cells again in G1 phase (Hartwell, 1974; reviewed in Nasmyth, 1993). The products of three S. cerevisiae G1 cyclin genes called CLN1, CLN2, and CLN3 are the principal limiting components for passage through START (Richardson et al., 1989; Wittenberg et al., 1990; Tyers et al., 1993). Transcription of CLN1 and CLN2 is activated in G1, and accumulation of their protein products to a critical threshold level by a positive feedback mechanism leads to activation of the p34CDC28 kinase and transition through START (Dirick and Nasmyth, 1991). The G1 cyclins are then degraded as a consequence of PEST motifs in their primary sequence that appear to result in rapid protein turnover (Rogers et al., 1986; Lew et al., 1991; reviewed in Reed, 1991).
The S. cerevisiae G1 cyclins are at least partially redundant, because yeast strains in which two of the three G1 cyclin genes are deleted and the third placed under the control of a galactose-regulated promoter show a galactose-dependent growth phenotype. Such strains have been used to identity Drosophila and human cDNA clones that rescue this conditional cln-deficient phenotype on glucose plates when the single yeast CLN gene present is repressed (Koff et al., 1991; Lahue et al., 1991; Leopold and O""Farrell, 1991; Lew et al., 1991; Xiong et al., 1991). Human cDNAs encoding three new classes of cyclins, C, D, and E, were identified by this means. Although these cyclins show only limited homology with the yeast CLN proteins, they have proved important for understanding controls that operate in mammalian cells during G1 and at the restriction point at the G1-to-S phase boundary (Pardee, 1989; Matsushime et al., 1992; Koff et al., 1992, 1993; Ando et al., 1993; Quelle et al., 1993; Tsai et al., 1993b). Cyclin E may act as a rate-limiting component at the G1-to-S phase boundary (Ohtsubo and Roberts, 1993; Wimmel et al., 1994), whereas the dependency of cyclin D levels on serum growth factors (Matsushime et al., 1991; Baldin et al., 1993; Sewing et al., 1993) suggests that cyclins of the D-type may form a link between these signals and cell cycle progression.
An important factor involved in the regulation of cell cycle progression in mammals is the retinoblastoma susceptibility gene encoding the retinoblastoma protein (Rb). Rb binds and inactivates the E2F family of transcription factors, and it is through this ability that Rb exerts most of its potential to restrain cell division in the G1-phase. E2F transcription factors are known to switch on cyclin E and S-phase genes and the rising levels off cyclin E and/or E2F lead to the onset of replication (Nevins, 1992, Johnson et al., 1993). The ability of Rb to inactivate E2F depends on its phosphorylation state. During most of G1, Rb is in a hypophosphorylated state, but in late G1 phase, phosphorylation of Rb is carried out by cyclin-dependent kinases particularly CDK4 complexed to its essential regulatory subunit, cyclin D (Pines, 1995) and CDK2 complexed to cyclin E (at the G1/S boundary) or cyclin A (in S phase). These multiple phosphorylations of Rb cause it to release E2F, which can then, ultimately promote transcription of the S-phase genes.
Plant cells were used in early studies of cell growth and division to define the discrete phases of the eukaryotic cell cycle (Howard and Pelc, 1953), but there is a paucity of data on molecular cell cycle control in plant systems. Plant cells that cease dividing in vivo due to dormancy, or in vitro due to nutrient starvation, arrest at principal control points in G1 and G2 (van""t Hof and Kovacs, 1972; Gould et al., 1981; reviewed in van""t Hof, 1985); this is in general agreement with the controls operating in other eukaryotic systems. Although mature plant cells may be found with either a G1 or a G2 DNA content (Evans and van""t Hof, 1974; Gould et al., 1981), the G1 population generally predominates. The G1 control point is found to be more stringent in cultured plant cells subjected to nitrogen starvation; these cells arrest exclusively in G1 phase (Gould et al., 1981). Strong analogies thus exist between the principal control point in G1 of the plant cell cycle, the START control in yeasts, and the restriction point of mammalian cells.
Antibodies or histone HI kinase assays have been used to indicate the presence and localization of active CDC2-related kinases in plant cells (John et al., 1989,1990, 1991; Mineyuki et al., 1991; Chiatante et al., 1993; Colasanti et al., 1993; reviewed in John et al., 1993), and cDNAs encoding functional homologs of CDC2 kinase have been isolated by reduced stringency hybridization or redundant polymerase chain reaction from a number of plant species, including pea (Feiler and Jacobs, 1990), alfalfa (Hirt et al., 1991, 1993), Arabidopsis (Ferreira et al., 1991; Hirayama et al., 1991), soybean (Miao et al., 1993), Antirrhinum (Fobert et al., 1994), and maize (Colasanti et al., 1991). A number of cDNA sequences encoding plant mitotic cyclins with A- or B-type characteristics or having mixed A- and B-type features have also been isolated from various species, including carrot (Hata et al., 1991), soybean (Hata et al., 1991), Arabidopsis (Hemerly et al., 1992; Day and Reddy, 1994), alfalfa (Hirt et al., 1992), Antirrhinum (Fobert et al., 1994), and maize (Renaudin et al., 1994).
Soni et al. (1995) identified a new family of three related cyclins in Arabidopsis by complementation of a yeast strain deficient in G1 cyclins. Individual members of this family showed tissue specific expression and are conserved in other plant species.
They form a distinctive group of plant cyclins and were named xcex4-type cyclins to indicate their similarities with mammalian D-type cyclins. The sequence relationships between xcex4 and D cyclins include the N-terminal sequence LxCxE. The leucine is preceded at position xe2x88x921 or xe2x88x922 by an amino acid with an acidic side chain (D, E). This motif was originally identified in certain viral oncoproteins and is strongly implicated in binding to the retinoblastoma protein. By analogy to mammalian cyclin D, these plant homologs may mediate growth and phytohormonal signals into the plant cell cycle. In this respect it was shown that, on restimulation of suspension-cultured cells, cyclin xcex43 was rapidly induced by the plant growth regulator cytokinin and cyclin xcex42 was induced by carbon source. Renaudin et al. (1996) defined the groups and nomenclature of plant cyclins and xcex4-cyclins are now called CycD cyclins.
Dahl et al. (1995) identified in alfalfa a cyclin (cycMs4) related to xcex43 in alfalfa.
Recently, Rb-like proteins were identified in plant. Both Xie et al. (1996) and Grafi et al. (1996) describe the isolation and preliminary characterization of an Rb homologue from maize.
Doerner et al. (1996) describe the ectopic expression of a B-type cyclin (cyc1At from Arabidopsis) under control of a promoter from the cdc2a gene, in Arabidopsis. The xe2x80x9ccdc2axe2x80x9d transgenic plants expressing the transgene strongly had a markedly increased root growth rate. Moreover, growth and development of lateral roots was accelerated following induction with indoieacetic acid in the transgenic plants relative to the control plants.
Hemerly et al. (1995) describe transgenic tobacco and Arabidopsis plants expressing wild type or dominant mutations of a kinase operating at mitosis (CDC2a). Plants constitutively overproducing the wild-type CDC2a or a mutant form predicted to accelerate the cell cycle did not exhibit a significantly altered development. A mutant CDC2a, expected to arrest the cell cycle, abolished cell division when expressed in Arabidopsis. Some tobacco plants constitutively producing the latter mutant kinase, were recovered. These plants contained considerably fewer but larger cells.
PCT patent publication xe2x80x9cWOxe2x80x9d 92/09685 describes a method for controlling plant cell growth comprising modulating the level of a cell cycle protein in a plant for a time and under conditions sufficient to control cell division. The preferred protein, identified in the examples, is a p34cdc2 kinase or the like operating at mitosis.
WO93/12239 describes plants with altered stature and other phenotypic effects, particularly precocious flowering and increased numbers of flowers by transformation of the plant genome with a cdc25 gene from a yeast such as Schizosaccharomyces pombe. 
WO97/47647 relates to the isolation and characterization of a plant DNA sequence coding for a retinoblastoma protein, the use thereof for the control of the growth in plant cells, plants and/or plant viruses as well as the use of vectors, plants, or animals or animal cells modified through manipulation of the control route based on the retinoblastoma protein of plants.
U.S. Pat. No. 5,514,571 discloses the use of cyclin D1 as a negative regulator of mammalian cell proliferation. Overexpression of cyclin D1 blocks mammalian cell growth, while blocking cyclin D1 expression promotes cell proliferation.
The invention provides a process to obtain a plant with altered growth characteristics or altered architecture, particularly plants with reduced or increased growth rate, plants which require less time to flower or plants with an increased number of flowers per plant, or plant with an increased size of an organ comprising the step of altering the level or the functional level of a cell-division controlling protein, capable of binding and/or phosphorylating an Rb-like protein, preferably a cell-division controlling protein comprising an LxCxE binding motif or related motif, preferably in the N-terminal part of the protein, particularly a D-type cyclin, within the cells of a plant.
Also provided is a process to obtain a plant with altered growth characteristics or altered architecture comprising the step of altering the level or functional level of the cell-division controlling protein by integrating a chimeric gene into the genome of the cells of the plant, comprising the following operably linked DNA fragments:
a) a plant expressible promoter region, particularly a CaMV35S promoter region,
b) a transcribed DNA region encoding an RNA or a protein, which when expressed, either increases or decreases the level or the functional level of the cell-division controlling protein; and optionally
c) a 3xe2x80x2 end formation and polyadenylation signal functional in plant cells.
In accordance with the invention, the transcribed DNA region encodes an antisense RNA, a ribozyme, or a sense RNA strand which when expressed reduces, inhibits or prevents the expression of a cell-division controlling protein particularly an endogenous D-type cyclin.
Further in accordance with the invention the transcribed DNA encodes a cell-division controlling protein capable of binding the pocket domain of an Rb-like protein, preferably a cell-division controlling protein comprising an LxCxE binding motif, more preferably a D-type cyclin, particularly a D-type cyclin from plants, more particularly a D-type cyclin is selected from group of Arabidopsis thaliana CYCD1, Arabidopsis thaliana CYCD2, Arabidopsis thaliana CYCD3, Nicotiana tabacum CYCD2;1, Nicotiana tabacum CYCD3;1, Nicotiana tabacum CYCD3;2, Helianthus tuberosus CYCD1;1, Zea mays CYCD2 and Helianthus tuberosus CYCD3;1.
Also in accordance with the invention the transcribed RNA encodes a protein or peptide which, when expressed, increases said functional level of said cell division controlling protein, particularly a protein or peptide selected from: a mutant D-type cyclin, a part of a D-type cyclin, a D-type cyclin which has a mutation in the cyclin box, a D2-type cyclin which has a substitution of amino acid 185 or amino acid 155, a D2-type cyclin which has mutation E185A or K155A, a D-type cyclin wherein the PEST sequences are removed, a D-type cyclin wherein the LxCxE binding motif has been changed or deleted, or a D-type cyclin wherein the C-residue from the LxCxE binding motif has been deleted.
It is also an object of the invention to provide such chimeric genes.
Further provided are plant cells, plants and seed thereof, comprising the chimeric genes of the invention and having altered growth characteristics and/or altered architecture.
Another object of the invention is to provide the use of a cell-division controlling protein, capable of binding the pocket domain of an Rb-like protein and/or capable of phosphorylating an Rb-like protein, particularly a cell-division controlling protein comprising an LxCxE binding motif in the N-terminal part of the protein, more particularly a D-type cyclin and genes encoding same, to alter the growth characteristics or architecture of a plant. The cell-division controlling protein is preferably encoded by a chimeric gene, integrated in the genome of the cells of a plant.
As used herein xe2x80x9carchitecturexe2x80x9d of a plant refers to the general morphology as defined by the relative sizes, positions and number of the several parts of a plant (i.e., organs such as but not limited to leaves, inflorescences, storage organs such as tubers, roots, stems, flowers, or parts of organs such as petals, sepals, anthers, stigma, style, petiole and the like). xe2x80x9cAltering the architecture of a plantxe2x80x9d thus refers to changes in the general morphology as the result of changing e.g., the number, size and position of organs or parts of organs. It is clear that altering either one organ or part of an organ or several organs or parts of organs, as described, will result in an altered plant architecture. This can be achieved by altering (i.e., enhancing or reducing) cell division activity in existing meristems and/or organ primordia or by creating de novo meristems.
As used herein, xe2x80x9cco-suppressionxe2x80x9d refers to the process of transcriptional and/or post-transcriptional suppression of RNA accumulation in a sequence specific manner, resulting in the suppresion of expression of homologous endogenous genes or transgenes. Suppressing the expression of an endogenous gene can be achieved by introduction of a transgene comprising a strong promoter operably linked to a DNA region whereby the resulting transcribed RNA is a sense RNA comprising a nucleotide sequence which is has at least 75%, preferably at least 80%, particularly at least 85%, more particularly at least 90%, especially at least 95% to the coding or transcribed DNA sequence (sense) of the gene whose expression is to be suppressed. Preferably, the transcribed DNA region does not code for a functional protein. Particularly, the transcribed region does not code for a protein.
As used herein, the term xe2x80x9cplant-expressible promoterxe2x80x9d means a promoter which is capable of driving transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, e.g., certain promoters of viral or bacterial origin such as the CaMV35S or the T-DNA gene promoters.
The term xe2x80x9cexpression of a genexe2x80x9d refers to the process wherein a DNA region under control of regulatory regions, particularly the promoter, is transcribed into an RNA which is biologically active i.e., which is either capable of interaction with another nucleic acid or protein or which is capable of being translated into a biologically active polypeptide or protein. A gene is said to encode an RNA when the end product of the expression of the gene is biologically active RNA, such as e.g., an antisense RNA or a ribozyme. A gene is said to encode a protein when the end product of the expression of the gene is a biologically active protein or polypeptide.
The term xe2x80x9cgenexe2x80x9d means any DNA fragment comprising a DNA region (the xe2x80x9ctranscribed DNA regionxe2x80x9d) that is transcribed into a RNA molecule (e.g., an mRNA) in a cell under control of suitable regulatory regions, e.g., a plant-expressible promoter. A gene may thus comprise several operably linked DNA fragments such as a promoter, a 5xe2x80x2 leader sequence, a coding region, and a 3xe2x80x2 region comprising a pqlyadenylation site. An endogenous plant gene is a gene which is naturally found in a plant species. A chimeric gene is any gene which is not normally found in a plant species or, alternatively, any gene in which the promoter is not associated in nature with part or all of the transcribed DNA region or with at least one other regulatory region of the gene.
This invention is based on the unexpected finding that chimeric genes comprising DNA encoding a cell-division controlling protein capable of binding an Rb-like protein, particularly a plant cyclin of the D-type, under control of a plant-expressible promoter could be stably integrated in the genome of plant cells, without deleterious effects, and furthermore that the increased expression of such a cell-division controlling protein, particularly a cyclin of the D-type, in the plant cells led to specific alterations in the growth rate and architecture of the resulting transformed plants.
Thus, the invention relates to modulating the level of expression or activity of functional cell-division controlling proteins, preferably in a stable mannner, within plant cells of a plant to alter the architecture or the growth rate or both of the transformed plant and its progeny. Conveniently, the level or functional level of cell-division controlling proteins is controlled genetically by altering the expression of genes encoding these cell-division controlling proteins. Increasing the level or functional level of a cell-division controlling protein genetically can be achieved e.g., by manipulating the copy number of the encoding gene(s), by altering the promoter region of the encoding genes or by manipulation of the genes regulating directly or indirectly the level of the expression of a cell-division controlling protein. Alternatively, the level of a cell-division controlling protein can be increased by stabilizing the mRNA encoding the cell-division controlling protein, or by stabilizing the cell-division controlling protein e.g., by removal of destruction motifs or so-called PEST sequences.
The functional level or activity of cell-division controlling protein can be increased by the decreasing the level of an antagonist or an inhibitor of the cell-divsion promoting protein, through techniques such as, but not limited to, providing the cell with a protein, such as an inactive cell-division controlling protein similar to the one whose functional level is to be increased, or part of a such a cell-division controlling protein, which is still capable of binding an inhibitor or other regulatory protein, or is still capable of binding to cyclin-dependent kinases.The functional level or activity of cell-division controlling protein can also be increased by alteration or mutation of the cell-division controlling protein to reduce or elimate binding of an antagonist or inhibitor of the activity of the cell division related protein.
Reducing the functional level of a cell-division controlling protein can be achieved e.g., by decreasing the mRNA pool encoding the cell-division controlling protein that is available for translation, through techniques such as, but not limited to, antisense RNA, ribozyme action or co-suppresion. Alternatively, the functional level of of cell-division controlling protein can be decreased by the increasing the level of an antagonist or an inhibitor of the cell-divsion promoting protein.
For the purpose of this invention, a xe2x80x9ccell-division controlling proteinxe2x80x9d is a polypeptide or protein which is required for the regulation of the progression through the cell cycle of a eukaryotic cell, preferably a plant cell, or a protein which can effect the entry of cells into the cell cycle or affect progression of cells through the cell cycle by direct interaction with a protein required for the regulation of progression through the cell cycle, or a polypeptide or protein which can assume an equivalent function but is not required for the regulation of the cell cycle.
Suitable cell-division controlling proteins are proteins capable of phosphorylating either alone or in combination with other proteins an Rb-like protein, preferably capable of phosphorylating an Rb-like protein in a plant cell in the G1-S transition phase, or are capable of binding the pocket domain of retinoblastoma-like (Rb-like) proteins, preferably proteins having an LxCxE binding motif comprised within the amino-acid sequence or a related motif such as LxSxE or FxCxE (binding motifs are represented in the one-letter amino acid code wherein x represents any amino-acid). Particularly preferred are cyclins which comprise the LxCxE binding motif (and/or related motif) in the N-terminal half of the protein, preferably within the first 50 amino acid residues, particularly within the first 30 amino acid residues, such as the cyclins of the D-type, particularly plant cyclins of the D-type, especially a cyclin from the group of Arabidopsis thaliana CYCD1, Arabidopsis thaliana CYCD2, Arabidopsis thaliana CYCD3, Nicotiana tabacum CYCD3;1, Nicotiana tabacum CYCD2;1, Nicotiana tabacum CYCD3;2, Helianthus tuberosus CYCD1;1, Zea mays CYCD2 and Helianthus tuberosus CYCD3;1 or a cyclin with essentially similar protein sequences.
The mentioned plant cyclins of the D-type are fully characterized by the amino acid sequence encoded by the DNA sequence of EMBL Accession No X83369 (hereinafter, may be referred to as SEQ ID NO: 27) from the nucleotide position 104 to the nucleotide position 1108 for Arabidopsis thaliana CYCD1, EMBL Accession No X83370 (hereinafter, may be referred to as SEQ ID NO:29) from the nucleotide position 195 to the nucleotide position 1346 for Arabidopsis thalina CYCD2, EMBL Accession No X83371 (hereinafter, may be referred to as SEQ ID NO:31) from the nucleotide position 266 to the nucleotide position 1396 for Arabidopsis thaliana CYCD3, the nucleotide sequence of SEQ ID NO: 1 from nucleotide position 182 to nucleotide position 1243 for Nicotiana tabacum CYCD2;1, the nucleotide sequence of SEQ ID NO:3 from nucleotide position 181 to nucleotide position 1299 for Nicotiana tabacum CYCD3;1, the nucleotide sequence of SEQ ID NO:5 from nucleotide position 198 to nucleotide position 1298 for Nicotiana tabacum CYCD3;2, the nucleotide sequence of SEQ ID NO:7 from nucleotide position 165 to nucleotide position 1109 for Helianthus tuberosus CYCD1;1, the nucleotide sequence of SEQ ID NO:9 from nucleotide position 48 to nucleotide position 1118 for Helianthus tuberosus CYCD3;1 and the nucleotide sequence of SEQ ID NO:26 from nucleotide position 316 to nucleotide position 1389 for Zea mays CYCD2.
It is thought that increasing, respectively decreasing, the level or the functional level or the activity of these cell-division controlling proteins accelerates, respectively delays, the transition of G1 to the S-phase in plant cells, or increases, respectively decreases, the proportion of actively dividing cells, by their interaction with Rb-like proteins affecting the ability of the Rb-like protein to inactivate certain transcription factors. It is further thought that expression of these cell-division controlling proteins interacting with Rb-like proteins effectively allows the cells to initiate division processes, whereas (over)expression of G2/mitotic cyclins (such as cyclins of the B-type or the cdc25 gene product) is in contrast expected to lead to faster progression through the G2/mitotic phases of cell cycles already started.
For the purpose of this invention xe2x80x9cRb-like proteinsxe2x80x9d are defined as proteins from the group of human Rbxe2x88x921 protein (Lee et al. 1987 ; Accession no P06400), human p107 (Ewen et al., 1991; Accession no L14812) and human p130 (Hannon et al.1993; Accession A49370), Drosophila RBF (Du et al., 1996; Accession no for DNA entry of the encoding gene X96975), mouse RB (Bemards et al. 1989; Accession no P13405) chicken RB (Boehmelt et al., 1994; Accession no X72218), Xenopus Rb (Destree et al. 1992; Accession A44879), ZmRb and Rb1 from Zea mays (Xie et al., 1996; Grafi et al. 1996; Accession numbers for DNA entry of the encoding genes: X98923; GenBank U52099) as well as any protein that has simultaneously at least 25-30% amino acid sequence similarity (identity) to at least three members of the above-mentioned group, and comprises the conserved cysteine residue located at position 706 of human Rb-1 or at equivalent positions in the other Rb-like proteins (see e.g., Xie et al. 1996).
Rb-like proteins are members of a small family known as xe2x80x9cpocket proteinsxe2x80x9d. This term is derived from a conserved bipartite domain, the so-called xe2x80x9cpocket domainxe2x80x9d, which is the binding site for several growth control proteins such as E2F family of transcription factors, D-type cyclins and viral oncoproteins. The A and B subdomains of the pocket domain are more conserved than the rest of the protein (xe2x88x9250-64% for the A and B subdomains) and are separated by a non-conserved spacer. Pocket domains are located between amino acids at positions 451 and 766 for human Rb, 321 to 811 for human p107, 438 to 962 for human p130, 445 to 758 for mouse RB, 441 to 758 for chicken RB, 440 to 767 for Xenopus Rb, 11 to 382 for com ZmRb, 89 to 540 for com Rb1.
For the purpose of the invention xe2x80x9cbinding to an Rb-like proteinxe2x80x9d or xe2x80x9cbinding to the pocket domain of an Rb-like proteinxe2x80x9d can be analyzed by either an in vitro assay or one of the in vivo assays, or a combination thereof. In the in vitro assay, the binding is analyzed between the protein in question which has been labelled by 35S-methionine, and a fusion protein of glutathione-S-transferase (GST) and the pocket domain of an Rb-like protein, such as the human Rb. The fusion to GST allows easy purification and fixation of the fusion protein on glutathione sepharose beads. The interaction between the assayed protein and the Rb-like protein is compared to the binding between the same protein and a fusion protein of GST and an Rb-like protein with a mutation in the conserved cysteine at a position equivalent to cysteine 706 in human Rb, such as human Rb C706F. Such an assay has been described e.g., by Dowdy et al. (1993) and Ewen et al. (1993). In a variant of this assay, the Rb-like protein can be expressed in baculovirus-infected insect cells (Dowdy et al., 1 993). In a further variant, both the Rb-like protein and Rb-binding protein can be co-expressed in insect cells, and association detected by gel-filtration or co-immunoprecipitation (O""Reilly et al., 1992).
An in vivo assay which can be used to determine the binding of a protein to the pocket domain of Rb-like proteins, is the yeast two-hybrid system (Fields and Song, 1989). This analysis relies on the ability to reconstitute a functional GAL4 activity from two separated GAL4 fusion proteins containing the DNA binding domain (GAL4BD) and the activation domain (GAL4AD) fused to a pocket domain of an Rb-like protein and the protein to be assayed respectively. Expression plasmids comprising chimeric genes encoding these fusion proteins are introduced into a yeast strain encoding appropriate GAL4 inducible markers, such as strain HF7c (Feilloter et al., 1994) containing GAL4-inducible HIS3 and LacZ markers, or strain Y190 (Harper et al., 1993). Proteins binding to the pocket domain of the Rb-like protein will allow growth in the absence of histidine. An example of a two-hybrid assay to demonstrate interaction of a protein with an Rb-like protein has been described by Durfee et al. (1993).
Preferably, suitable control experiments should be included, such as separate introduction into the same yeast strain of the expression plasmids, or introduction of expression plasmids encoding fusion proteins containing the DNA binding domain (GAL4BD) and the activation domain (GAL4AD) fused to a mutated pocket domain of an Rb-like protein, preferably mutated at the C706 or equivalent positions and the protein to be assayed respectively.
An alternative in vitro assay to determine the binding of a protein to the pocket domain of Rb-like proteins comprises transient expression of both proteins in plant cells, preferably tobacco protoplasts, and immunoprecipitation using an antibody directed against one of the two proteins to measure co-precipitation of the other protein.
For the purpose of the invention xe2x80x9cphosphorylating an Rb-like proteinxe2x80x9d can be analyzed by an in vitro assay relying on the use of gamma 32P-labeled adenosine-triphosphate to monitor the capacity of a protein (or a combination of proteins such as cyclins and cyclin dependent kinases) to transfer the labeled phosphate group to a target protein, as known in the art.
For the purpose of the invention xe2x80x9ccyclinxe2x80x9d can be defined as a regulatory protein, comprising a protein domain of about 100 amino acids known as the xe2x80x9ccyclin boxxe2x80x9d. The cyclin box is the binding site for cyclin-dependent kinases, allowing the cyclin to exert its regulatory effect on the kinase activity of the CDKs.
A cyclin box can be identified by comparing the amino acid sequence of the protein with known cyclin boxes, such as the amino acid sequence between positions 81-186 of CYCD1 from Arabidopsis thaliana, between positions 96-201 of CYCD2 from Arabidopsis thaliana, between positions 86-191 of CYCD3 from Arabidopsis thaliana, the cyclin boxes described by Renaudin et al. (1994; 1996), by Soni et al. (1995), and by Hemerly et al. (1992). An amino acid sequence identified as a cyclin box on the basis of sequence comparison should posses at least the five conserved residues required for cyclin activity R(97), D(126), L (144), K(155), E(185) (indicated positions are from the sequence of CYCD2 from Arabidopsis thaliana) at equivalent positions. (see e.g., Soni et al. (1995) and Renaudin et al. (1996).
D-type cyclins (cyclin D or CycD) are cyclins that are characterized by the presence of additional characteristic sequences, such as the LxCxE motif or related motifs for binding Rb-like proteins, which is found within the N-terminal part of the protein, preferably located between the N-terminus and the cyclin box, particularly within the first 50 amino acids, more particularly within the first 30 amino acids of the initiating methionine-residue. Preferably, the leucine of the binding motif is preceded at position xe2x88x921 or xe2x88x922 by an amino acid with an acidic side chain (D, E). Altemative binding motifs such as LxSxE or FxCxE can be found. Indeed, Phelps et al. (1992) have identified that mutating the binding motif LxCxE in human papillomavirus E7 to LxSxE does not affect the ability of the protein to bind Rb-like proteins. Three groups of D-type cyclins have been identified on the basis of sequence homology: CycD1 (comprising Arabidopsis thaliana CycD1 and Helianthus tuberosus CYCD1;1) CycD2 (comprising Arabidopsis thaliana CYCD2, Nicotiana tabacum CYCD2;1, Zea mays CYCD2), CycD3 (comprising Arabidopsis thaliana CYCD3, Nicotiana tabacum CYCD3;1, Nicotiana tabacum CYCD3;2, and Helianthus tuberosps CYCD3;1).
Nomenclature and consensus sequences for the different types and groups of plant cyclins, including cyclins of the D-type, have been described by Renaudin et al. (1996) and can be used to classify new cyclins based on their amino acid sequence.
For the purpose of the invention, the cell-division controlling proteins can be provided to the cells either directly, e.g., by electroporation of the protoplasts in the presence of the cell-division controlling proteins, or indirectly, by transforming the plant cells with a plant-expressible chimeric gene encoding the protein to be tested either transiently, or stably integrated in the genome of the protoplasts.
In one aspect of the invention the level or the functional level of the cell-division controlling protein, capable of phosphorylating an RB-like protein or binding the pocket domain of an Rb-like proteins, is increased, to obtain a plant with altered growth rate or architecture, by integrating a chimeric gene into the genome of the cells of the plant, comprising the following operably linked DNA fragments:
a) a plant-expressible promoter region, particularly a CaMV35S promoter region,
b) a transcribed DNA region encoding a protein, which when expressed, increases the level or the functional level of the cell-division controlling protein; and optionally
c) a 3xe2x80x2 end formation and polyadenylation signal functional in plant cells.
In a preferred embodiment of the invention, the expression level of cyclin D is increased by introduction into the genome of a plant cell, a chimeric gene comprising a transcribed DNA region encoding a cyclin D, under control of a plant-expressible promoter. The transcribed DNA region preferably comprises a nucleotide sequence selected from the nucleotide sequence of EMBL Accession No X83369 (SEQ ID NO:27) from the nucleotide position 104 to the nucleotide position 1108, the nucleotide sequence of EMBL Accession No X83370 (SEQ ID NO:29) from the nucleotide position 195 to the nucleotide position 1346, the nucleotide sequence of EMBL Accession No X83371 (SEQ ID NO:31) from the nucleotide position 266 to the nucleotide position 1396, the nucleotide sequence of SEQ ID NO:1 from nucleotide position 182 to nucleotide position 1243, the nucleotide sequence of SEQ ID NO:3 from nucleotide position 181 to nucleotide position 1299, the nucleotide sequence of SEQ ID NO:5 from nucleotide position 198 to nucleotide position 1298, the nucleotide sequence of SEQ ID NO:7 from nucleotide position 165 to nucleotide position 1109, the nucleotide sequence of SEQ ID NO:9 from nucleotide position 48 to nucleotide position 1118 or the nucleotide sequence of SEQ ID NO:26 from nucleotide position 316 to nucleotide position 1389 for Zea mays CYCD2.
In a particularly preferred embodiment the expression level of a cyclin of the CycD2 type is altered (i.e., increased) by introduction into the genome of a plant cell, of a xe2x80x9cchimeric cycD2 genexe2x80x9d comprising a transcribed DNA region encocding a cyclin of the CycD2 type, under control of a plant-expressible promoter, preferably a constitutive promoter, particularly a CaMV35S promoter, such as the chimeric cycD2 gene of plasmid pCEC1, in order to alter the morphology, architecture and growth characteristics of the transgenic plant, particularly to increase the vegetative growth of the transgenic plant, more particularly to alter the growth rate of the transgenic plant.
For the purpose of the invention, xe2x80x9cincreasexe2x80x9d or xe2x80x9cdecreasexe2x80x9d of a measurable phenotypic trait is quantified as the difference between the mean of the measurements pertinent to the description of that trait in different plants of one transgenic plant line, and the mean of the measurements of that trait in wild type plants, divided by the mean of the measurements of that trait in wild type plants, expressed in percentage, whereby transgenic and control (wild type) plants are grown under the same conditions of nutrient supply, light, moisture, temperature and the like, preferably under standardized conditions. Prefered levels of increase or decrease are statistically significant, preferably at the 0.05 confidence level, particularly at the 0.01 confidence level, e.g., by one way variance analysis (e.g., as described in Statistical Methods by Snedecor and Cochran).
Increase of the vegetative growth of a transgenic plant is preferably monitored by measuring the increase in dry weight during the growth period. The mean increase of dry weight is defined as the difference in mean dry weight of transgenic plants and wild type plants multiplied by 100 and divided by the mean dry weight of wild type plants. Typical increases in dry weight, particularly early in growth period, by introduction of the chimeric cycD2 genes of the invention range from at least about 39% to about 350%, particularly from about 68% to about 150%.
It is clear that increases in dry weight resulting from introduction of the chimeric genes of the invention may vary, depending on the plant species or chimeric genes used, and any significant increase in dry weight in transgenic plants is encompassed by the invention, particularly a dry weight of at least about 1.4 times to at least about 4.5 times the dry weight in untransformed control plants, particularly of at least about 1.8 times to at least about 2.7 times the dry weight in untransformed control plants. In any case, the mean dry weight of the transgenic plants is statistically significantly different from the mean dry weight of the untransformed plants.
Increase in the vegetative growth of a transgenic plant can also be determined by comparing the number of leaves visible on the transgenic plants and the control wild-type plants at any given, point in time. The difference in number of leaves of transgenic plants in the middle of the growth period is expected to be at least about 1.1 to at least about 3 times, particularly at least about 1.5 to at least about 2 times the leaf number in untransformed plants.
Increase of the vegetative growth of a transgenic plant can also be monitored by measuring the height of the stem (measured from soil level to the top of growing point) during the growth period. The mean increase of the stem height is defined as the difference in mean stem height of transgenic plants and wild type plants multiplied by 100 and divided by the mean height of wild type plants. Typical increases in stem height by introduction of the chimeric cycD2 genes of the invention range from at least about 65% early during growth, over at least about 20-30% in the middle of the growing period, to at least about 10% by the time of flowering, but may be as high as about 120% to about 190% early during growth, as high as about 40-50% to about 75% in the middle of the growing period, and as high as about 15-20% at the end of the flowering stage.
It is clear that increases in stem height resulting from introduction of the chimeric genes of the invention may vary, depending on the plant species or chimeric genes used, and any significant increase in stem height in transgenic plants is encompassed by the invention, particularly stem height of at least about 1.1 times to at least about 3 times the stem height in untransformed control plants, particularly of at least about 1.5 times to at least about 2 times the stem height in untransformed control plants.
The difference in stem height between transgenic and control plants diminishes as growth progresses, because the growth rate slows down in plants that are flowering. The terminal height of a transgenic plant, may thus be similar to the terminal height of a non-transgenic plant.
The transgenic plants comprising the chimeric cycD2 genes of the invention have an increased growth rate, when compared with untransformed plants, resulting in a reduced time required to reach a given dry weight or stem height. xe2x80x9cGrowth ratexe2x80x9d as used herein, refers to the increase in size of a plant or part of plant per day, particularly to increase in stem height per day, and can be calculated as the difference between the size of a plant or part of a plant at the start and end of a period comprising a number of days, particularly 6 to 8 days, divided by the number of days. Increase in growth rate is preferably expressed according to the general definition of increase of a measurable phenotype, but can also be expressed as the ratio between the growth rate of the transgenic plants, versus the growth rate of the untransformed control plants, during the same period, under the same conditions.
As mentioned before the increase in growth rate resulting from introduction of the chimeric genes, particularly the chimeric cycD2 genes of the invention may vary, depending on the plant species or the chimeric genes used, and any significant increase in growth rate in transgenic plants is encompassed by the invention, particularly increase in growth rate ranging from about 4% to about 85%, more particularly from about 20% to about 60%, especially from about 30% to about 50%.
Increase of the vegetative growth of a transgenic plant can also be monitored by measuring the length or the size of the largest leaf at different time points during the growth period whilst the leaves are still expanding. This measurable phenotype is a measure of the increased maturity of the transgenic plants. The mean increase of the length of the largest leaf (defined as the difference between mean length of the largest leaf of transgenic plants and wild type plants multiplied by 100 and divided by the mean length of the largest leaf of wild type plants) obtained by introduction of the chimeric genes of the invention ranges from about 7 to 31% (mean about 17%) early during growth, to about 3-14% (mean about 7%) in the middle of the growing period.
Again, these increases in the size of the largest leaf, resulting from introduction of the chimeric genes of the invention, may vary, depending on the plant species or chimeric gene used, and any significant increase in leaf growth or size in transgenic plants is encompassed by the invention.
As another object of the invention, the chimeric cell-division controlling gene, particularly the chimeric cycD2 genes, can also be introduced in plants to increase the root development, particularly to increase the mean root length. In general, the increase in root development, is parallel to the increase in the vegetative part above the ground (stem, leaves, flowers) and may range from about 40% to about 70%, but again these increases may vary depending on the plant species or chimeric gene used, and any significant increase, particularly statistically significant increase in root development is encompassed by the invention.
As yet another object of the invention, the chimeric cell-division controlling gene, particularly the chimeric cycD2 genes, can also be introduced in plants to increase the size as well as the number of flowers, particularly the number of fertilized flowers, and the number of fertilized ovules in each flower. As a result of the increase in the number of fertilized flowers, and the number of fertilized ovules in each flower (generally leading to a greater number of seeds per plant), it is clear that; also an increase in seed yield per plant can be obtained. It is clear that the increase in the number of flowers and ovules per flower, as well as the increase in seed yield can vary, depending on the plant species transformed with the chimeric cell-division controlling genes of the invention or the chimeric genes used. Typical increases in flower size resulting from the introduction of a chimeric gene comprising a CycD2 encoding DNA region under control of a CaMV35S promoter range from at least about 4% to at least about 30%, particularly at least about 10% to at least about 20%. Typical increases in the number of flowers range from about at least 20% to at least about 50%, particularly from about 24% to about 45% while increases in the number of seeds/plants (expressed on a weight basis) are in a range from at least about 5% to at least about 55%, particularly from at least about 10% to at least about 30%, more particularly about 25%.
In still another embodiment of the invention, the chimeric cell-division controlling gene, particularly the chimeric cycD2 genes, can also be introduced in plants or their seeds to accelerate germination. It has been found that transgenic seeds comprising the chimeric cycD2 genes of the invention can germinate at least between about 8 to about 16 hrs faster than wild type controls.
Moreover, the mentioned chimeric genes can also be introduced in plants to decrease the mean number of days required to reach the development of an inflorescence, thus effectively reducing the time required to start flowering. Transgenic plants comprising the chimeric cycD2 genes of the invention thus reach maturity, particularly the flowering stage, earlier, but have the normal size of a flowering plant. The actual reduction in time required to reach the flowering stage may depend on the plant species or chimeric genes used. Typically, transgenic plants harboring the chimeric gene comprising a CycD2 encoding DNA region under control of a CaMV35S promoter exhibit a reduction in the time required to flower of at least about 3% to 11-12%, particularly at least about 4% to 7%.
In another particularly preferred embodiment, a chimeric gene comprising a CycD3 encoding transcribed DNA region under control of a plant-expressible promoter, preferably a constitutive promoter, particularly a CaMV35S promoter, such as a chimeric gene comprising the nucleotide sequence of the chimeric cycD3 gene of pCRK9, is introduced into a plant cell to obtain transgenic plants with altered morphological traits or architecture, particularly with altered size of specific plant parts or organs, more particularly with altered flower size and morphology such as flowers with elongated and/or enlarged petals. Transgenic plants transformed with a chimeric gene comprising a CycD3 encoding DNA region under control of a plant-expressible promoter (and the progeny thereof) exhibit an increase in the flower size of about 31% to about 44%. Moreover these transgenic plants also flower later than wild type plants, corresponding to an increase in flowering time of about 5% to about 20%, particularly about 8% to about 16%.
In another embodiment of the invention the functional level of the cell-division controlling protein, capable of phosphorylating an RB-like protein or binding the pocket domain of an Rb-like proteins, particularly of the D-type cyclin is increased, to obtain a plant with altered growth rate or architecture, by integrating a chimeric gene into the genome of the cells of the plant, comprising the following operably linked DNA fragments:
a) a plant-expressible promoter region, particularly a CaMV35S promoter region,
b) a transcribed DNA region encoding a protein, which when expressed, increases the functional level of a cell-division controlling protein, preferably encoding a mutant cell-division controlling protein or part of a mutant cell-division controlling protein, more preferably encoding a mutant D-type cyclin or part of a D-type cyclin, particularly encoding a D-type cyclin which has a mutation in cyclin box (quite particularly a substitution of amino acid 185 or amino acid 155 of a D2-type cyclin, especially E185A or K155A), or a D-type cyclin wherein the PEST sequences are removed, particularly which has been C-terminally deleted to remove the PEST sequences, or a D-type cyclin wherein the LxCxE binding motif has been changed or deleted, particularly wherein the C-residue from the LxCxE binding motif has been deleted; and optionally
c) a 3xe2x80x2 end formation and polyadenylation signal functional in plant cells.
Although not intending to limit the invention to a mode of action, it is believed that the mutant cell-division controlling proteins exert their effects by sequestering inhibitors or antagonists of the normal functional cell-division controlling proteins.
It is clear from this description that chimeric genes comprising a transcribed DNA region encoding other cyclins of the D-type, particularly plant-derived cyclins of the CycD group, may be used to obtain similar effects. These genes can be obtained from other plant species or varieties, by different methods including hybridization using the available CycD1, CycD2 or CycD3 encoding DNAs as probes and hybridization conditions with reduced stringency, or polymerase chain reaction based methods using oligonucleotides based on the available nucleotide sequences of D-type cyclins, preferably oligonucleotides having a nucleotide sequence corresponding to the sequences encoding the consensus amino acid sequences, particularly oligonucleotides having a nucleotide sequence corresponding to the sequences encoding conserved amino acid sequences within the cyclin box for each group of cyclins. These conserved amino acid sequences can be deduced from available aligned DNA encoding such amino acid sequences. A particularly preferred combination of oligonucleotides for PCR amplification of plant cyclins of the D1 type is an oligonucleotide selected from the group of oligonucleotides having the DNA sequence of SEQ ID NO:12 SEQ ID NO:13 or SEQ ID NO:14 and an oligonucleotide selected from the group of oligonucleotides having the DNA sequence of SEQ ID NO:15 or SEQ ID NO:16.
A particularly preferred combination of oligonucleotides for PCR amplification of plant cyclins of the D2 type is an oligonucleotide selected from the group of oligonucleotides having the DNA sequence of SEQ ID NO:17 or SEQ ID NO:18 and an oligonucleotide selected from the group of oligonucleotides having the DNA sequence of SEQ ID NO:19 or SEQ ID NO:20. A particularly preferred combination of oligonucleotides for PCR amplification of plant cyclins of the D3 type is an oligonucleotide selected from the group of oligonucleotides having the DNA sequence of SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23 and an oligonucleotide selected from the group of oligonucleotides having the DNA sequence of SEQ ID NO:24 or SEQ ID NO:25. The amplified DNA fragment is then used to screen a cDNA or genomic library (under stringent conditions) to isolate full length clones.
Alternatively, additional genes encoding plant-derived cyclins can be obtained by techniques such as, but not limited to, functional complementation of conditional G1-S cyclin deficient yeast strains, as described by Soni et al. (1995) and Dahl et al. (1995) or by using the yeast two-hybrid system (Fields and Song, 1989) to isolate DNA sequences encoding cyclins binding to the pocket domain of Rb-like proteins as described supra.
It is further known that some plants contain more than one gene encoding a D-type cyclin of the same subgroup (e.g., tobacco contains at least two genes of the CycD3 subgroup) and it is clear that these variants can be used within the scope of the invention.
Moreover D-type cyclins which have an amino acid sequence which is essentially similar to the ones disclosed in this invention, such as mutant D-type cyclins, can be used to the same effect. With regard to xe2x80x9camino acid sequencesxe2x80x9d, essentially similar means that when the two relevant sequences are aligned, the percent sequence identity i.e., the number of positions with identical amino acid residues divided by the number of residues in the shorter of the two sequences- is higher than 80%, preferably higher than 90%. The alignment of the two amino acid sequences is performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983) using a window-size of 20 amino acids, a word length of 2 amino acids, and a gap penalty of 4. Computer-assisted analysis and interpretation of sequence data including sequence alignment as described above, can be conveniently performed using the programs of the lntelligenetics(trademark) Suite (lntelligenetics Inc. CA).
It is clear that any DNA sequence encoding a cell-division controlling protein, particularly a D-type cyclin, can be used to construct the chimeric cell-division controlling genes of the invention, especially DNA sequences which are partly or completely synthesized by man.
It is also clear that other plant-expressible promoters, particularly constitutive promoters, such as the the opine synthase promoters of the Agrobacterium Ti- or Ri-plasmids, particularly a nopaline synthase promoter can be used to obtain similar effects. Moreover, in the light of the existence of variant forms of the CaMV35S promoter, as known by the skilled artisan, the object of the invention can be equally be achieved by employing these alternative CaMV35S promoters, such as those described by Hull and Howell, Virology, 86, pg. 482 (1978).
It is a further object of the invention to provide plants with altered morphology or architecture, restricted to specific organs or tissues by using tissue-specific or organ-specific promoters to control the expression of the DNA encoding a cell-division controlling protein, particularly a cyclin of the D-type. Such tissue-specific or organ-specific promoters are well known in the art and include but are not limited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et al., 1996), stem-specific promoters (Keller et al., 1988), leaf specific promoters (Hudspeth et al., 1989), mesophyl-specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al., 1989), tuber-specific promoters (Keil et al., 1989), vascular tissue specific promoters (Peleman et al., 1989), meristem specific promoters (such as the promoter of the SHOOTMERISTEMLESS (STM) gene, Long et al., 1996), primordia specific promoter (such as the promoter of the Antirrhinum CycD3a gene, Doonan et al., 1998) and the like.
In another embodiment of the invention, the expression of a chimeric gene encoding a cell-division controlling protein can be controlled at will by the application of an appropriate chemical inducer, by operably linking the DNA region coding for the cell-division controlling protein to a promoter whose expression is induced by a chemical compound, such as the promoter of the gene disclosed in European Patent publication xe2x80x9cEPxe2x80x9d 0332104, or the promoter of the gene disclosed in WO90/08826.
In yet another embodiment of the invention, the expression of a chimeric gene encoding a cell-division controlling protein can be controlled by use of site-specific recombinases and their corresponding cis-acting sequences, e.g., by inserting between the plant-expressible promoter and the transcribed region encoding the cell-division controlling protein, an unrelated nucleotide sequence (preferably with transcriptional and/or translational termination signals) flanked by the cis-acting sequences recognized by a site-specific recombinase (e.g., lox or FRT sites); providing the plant cells comprising this chimeric gene with the site-specific recombinase (e.g., Cre or FLP) so that the inserted unrelated nucleotide sequence is eliminated by recombination, thus allowing the chimeric cell division controlling gene to be expressed.
It is thought that the morphological alterations obtained by increased expression of cell-division controlling proteins, particularly D-type cyclins in plants due to the introduction of a chimeric gene comprising a DNA region encoding a cell-division controlling protein, particularly a D-type cyclin under control of a plant expressible promoter, can be enhanced, by removal, adaptation or inactivation of PEST sequences. PEST sequences are amino acid sequences which are rich in proline, glutamate or aspartate and serine or threonine, located between positively charged flanking residues, which are involved in rapid turnover of the protein comprising such sequences (Tyers et al., 1992; Cross, 1988; Wittenberg and Reed, 1988; Salama et al., 1994). Removal of these PEST sequences in yeast cyclins stabilizes the cyclins in vivo (Pines, 1995). PEST regions can be identified by computer analysis, using software packages such as PESTFIND (Rogers et al., 1986; Rechsteiner, 1990). Mutation of a DNA encoding cell-division controlling protein with altered PEST sequences is well within the reach of the skilled artisan using methods such as described e.g., by Sambrook et al. (1989)
It is further expected that the quantitative effects of phenotypic alterations can be modulated -i.e., enhanced or repressed- by expression of endogenous cell-division controlling encoding chimeric genes, particularly endogenous CycD encoding chimeric genes as an alternative to using heterologous genes encoding similar proteins from other plants. Preferably, heterologous genes are used, particularly heterologous genes encoding similar proteins with less than about 65% , preferably less than about 75%, more preferably less than about 65% amino acid sequence identity to the endogenous cell division controlling protein.
In another aspect of this invention, the morphology of plants can be altered by decreasing expression of a functional cell-division controlling protein, particularly a D-type cyclin. This can be achieved using e.g., antisense-RNA, ribozyme, or co-suppresion techniques. To this end, a chimeric gene comprising a transcribed DNA region which is transcribed into an RNA, the production of which reduces, inhibits or prevents the expression of a cell-division controlling protein, particularly a D-type cyclin within the plant cells is introduced in the plant cells, particularly stably integrated in the genome of the plant cells.
In one embodiment of this aspect, the transcribed DNA region of the chimeric gene encodes an antisense RNA which is complementary to at least part of a sense mRNA encoding a cell-division controlling protein, particularly a D-type cyclin. The antisense RNA thus comprises a region which is complementary to a part of the sense mRNA preferably to a continuous stretch thereof of at least 50 bases in length, particularly of at least between 100 and 1000 bases in length. The antisense RNA can be complementary to any part of the mRNA sequence: it may be complementary to the sequence proximal to the 5xe2x80x2 end or capping site, to part or all of the leader region, to an intron or exon region (or to a region bridging an exon and intron) of the sense pre-mRNA, to the region bridging the noncoding and coding region, to all or part of the coding region including the 3xe2x80x2 end of the coding region, and/or to all or part of the 3xe2x80x2 or trailer region. The sequence similarity between the antisense RNA and the complement of the sense RNA encoding a cell-division controlling protein, should be in the range of at least about 75% to about 100%.
In another embodiment of this aspect, the transcribed DNA region of the chimeric gene encodes a specific RNA enzyme or so-called ribozyme (see e.g., WO 89/05852) capable of highly specific cleavage of the sense mRNA encoding a cell-division controlling protein, particularly a D-type cyclin.
In yet another embodiment, the level of a functional cell-division controlling protein, particularly a D-type cyclin can be decreased by the expression of chimeric gene comprising a DNA region encoding a protein or polypeptide which when expressed reduces the level of a cell-division controlling protein, particularly a D-type cyclin, or inhibits the cell division controlling protein, particularly the D-type cyclin, to exert its function within the plant cells. Preferably, the chimeric gene encodes an antibody that binds to a cell-division controlling protein, particularly a D-type cyclin.
Decreasing the level or the functional level of a cell-division controlling protein, particularly a D-type cyclin within the cells of a transgenic plant, comprising the chimeric genes of this embodiment of the invention, results in altered architecture, particularly in a decreased stem height, a decrease of the growth rate or a delaying in the flowering of the transgenic plants when compared to untransformed plants, grown under the same conditions. The effect obtained might vary, depending on the plant species or chimeric genes used, and any effect on architecture and/or growth rate, particularly a decrease in stem height or growth rate, or an increase in the time required to develop an inflorescence, is encompassed by the invention.
The decrease in growth rate due to decreasing the level of a cell-division controlling protein, preferably a D-type cyclin, particularly a CYCD2 type cyclin, ranges from about 30% to about 60%, particularly from about 35% to about 50%.
The decrease in stem height due to decreasing the level of a cell-division controlling protein, preferably a D-type cyclin, particularly a CYCD2 type cyclin, ranges from about 10% to about 60%, particularly from about 30% to about 50%, more particularly around 40%.
The increase in flowering time due to decreasing the level of a cell-division controlling protein, preferably a D-type cyclin, particularly a D2 type cyclin, ranges from about 10% to about 40%, particularly from about 15% to about 38%.
The chimeric cell-division controlling gene may include further regulatory or other sequences, such as leader sequences [e.g., cab22L leader from Petunia or the omega leader from TMV (Gallie et al., 1987)], 3xe2x80x2 transcription termination and polyadenylation signals (e.g., of the octopine synthase gene [De Greve et al., 1982)], of the nopaline synthase gene [Depicker et al., 1982] or of the T-DNA gene 7 [Velten and Schell, 1985] and the like [Guerineau et al., 1991; Proudfoot, 1991; Safacon et al., 1991; Mogen et al., 1990; Munroe et al., 1990; Ballas et al., 1989; Joshi et al.,1987], plant translation initiation consensus sequences [Joshi, 1987], introns [Luehrsen and Walbot, 1991] and the like, operably linked to the nucleotide sequence of the chimeric cell-division controlling gene.
Preferably, the recombinant DNA comprising the chimeric cell-division controlling gene is accompanied by a chimeric marker gene. The chimerc marker gene can comprise a marker DNA that is operably linked at its 5xe2x80x2 end to a plant-expressible promoter, preferably a constitutive promoter, such as the CaMV 35S promoter, or a light inducible promoter such as the promoter of the gene encoding the small subunit of Rubisco; and operably linked at its 3xe2x80x2 end to suitable plant transcription 3xe2x80x2 end formation and polyadenylation signals. It is expected that the choice of the marker DNA is not critical, and any suitable marker DNA can be used. For example, a marker DNA can encode a protein that provides a distinguishable color to the transformed plant cell, such as the A1 gene (Meyer et al., 1987), can provide herbicide resistance to the transformed plant cell, such as the bar gene, encoding resistance to phosphinothricin (EP 0,242,246), or can provided antibiotic resistance to the transformed cells, such as the aac(6xe2x80x2) gene, encoding resistance to gentamycin (WO94/01560).
Although it is clear that the invention can be applied essentially to all plant species and varieties, the invention will be especially suited to alter the architecture or to increase the growth rate of plants with a commercial value. It is expected that the enhancements in vegetative growth will be most pronounced in plants which have not undergone extensive breeding and selection for fast vegetative growth. The invention will be particularly relevant for plant which are grown in greenhouse, particularly to reduce the time required for greenhouse plants to reach the desired developmental stage, such as but not limited to flowering, fruit setting or seed setting. The invention will further be relevant to enhance the growth rate of trees, particularly softwood trees such as pine, poplar, Eucalyptus trees and the like. Another important application of the invention encompasses the expansion of effective area wherein plants can be cultivated by reduction of the time required to reach the economically important developmental stage. Particularly preferred plants to which the invention can be applied are corn, oil seed rape, linseed, wheat, grasses, alfalfa, legumes, a brassica vegetable, tomato, lettuce, rice, barley, potato, tobacco, sugar beet, sunflower, and ornamental plants such as carnation, chrysanthemum, roses, tulips and the like.
A recombinant DNA comprising a chimeric cell-division controlling gene can be stably incorporated in the nuclear genome of a cell of a plant. Gene transfer can be carried out with a vector that is a disarmed Ti-plasmid, comprising a chimeric gene of the invention, and carried by Agrobacterium. This transformation can be carried out using the procedures described, for example, in EP 0,116,718. Alternatively, any type of vector can be used to transform the plant cell, applying methods such as direct gene transfer (as described, for example, in EP 0,233,247), pollen-mediated transformation (as described, for example, in EP 0,270,356, WO85/01856 and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (as described, for example, in EP 0,067,553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (as described, for example, in U.S. Pat. No. 4,536,475), and the like.
Other methods, such as microprojectile bombardment as described, for corn by Fromm et al. (1990) and Gordon-Kamm et al. (1990), are suitable as well. Cells of monocotyledonous plants, such as the major cereals, can also be transformed using wounded and/or enzyme-degraded compact embryogenic tissue capable of forming compact embryogenic callus, or wounded and/or degraded immmature embryos as described in WO92/09696. The resulting transformed plant cell can then be used to regenerate a transformed plant in a conventional manner.
The obtained transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the chimeric cell-division controlling gene of the invention in other varieties of the same or related plant species. Seeds obtained from the transformed plants contain the chimeric cell-division controlling gene of the invention as a stable genomic insert.
The following non-limiting Examples describe the construction of chimeric cell-division controlling genes and the use of such genes for the modification of the architecture and growth rate of plants. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.
Throughout the description and Examples, reference is made to the following sequences:
SEQ ID NO:1 cDNA encoding Nicotiana tabacum CYCD2;1
SEQ ID NO:3 cDNA encoding Nicotiana tabacum CYCD3;1
SEQ ID NO:5 cDNA encoding Nicotiana tabacum CYCD3;2
SEQ ID NO:7 cDNA encoding Helianthus tuberosus CYCD1;1
SEQ ID NO:9 cDNA encoding Helianthus tuberosus CYCD3;1
SEQ ID NO:11 T-DNA of pGSV5
SEQ ID NO:12 PCR primer 1
SEQ ID NO:13 PCR primer 2
SEQ ID NO:14 PCR primer 3
SEQ ID NO:15 PCR primer 4
SEQ ID NO:16 PCR primer 5
SEQ ID NO:17 PCR primer 6
SEQ ID NO:18 PCR primer 7
SEQ ID NO:19 PCR primer 8
SEQ ID NO:20 PCR primer 9
SEQ ID NO:21 PCR primer 10
SEQ ID NO:22 PCR primer 11
SEQ ID NO:23 PCR primer 12
SEQ ID NO:24 PCR primer 13
SEQ ID NO:25 PCR primer 14
SEQ ID NO:26 cDNA encoding Zea mays CYCD2
Plasmids pCEC1 and pCRK9 have been deposited at the
Belgian Coordinated Collections of Microorganisms (BCCM)
Laboratorium voor Moleculaire Biologie-Plasmidecollectie (LMBP)
Universiteit Gent
K. L. Ledeganckstraat 35
B-9000 Gent, Belgium
on Mar. 11, 1997 and have been attributed the following deposition numbers:
MC1061(pCEC1): BCCM/LMBP3657
DH5xcex1 (pCRK9): BCCM/LMBP3656
Plasmids pBlueScript-ZM18 has been deposited at the
Belgian Coordinated Collections of Microorganisms (BCCM)
Laboratorium voor Moleculaire Biologie-Plasmidecollectie (LMBP)
Universiteit Gent
K. L. Ledeganckstraat 35
B-9000 Gent, Belgium
on Mar. 19, 1998 under the deposit number BCCM/LMBP 3866.